Kegg Pathway: Calcium signaling pathway

PLoS One. 2009; 4(11): e7877
Dunn TA, Storm DR, Feller MB

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, Calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between Calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular Calcium concentration and PKA activity was used to assay the role of different classes of Calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced Calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant Calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in Calcium-dependent activation of the cAMP/PKA cascade in neurons.

Am J Physiol Cell Physiol. 2009 Nov 18;
Xing J, Strange K

The C. elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+) selective TRPM channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCgamma, is also required for IP3-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP3 receptor activity. We demonstrate here that PLCgamma via hydrolysis of PI(4,5)P2 (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCgamma activity by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 muM PIP(2) inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC50 value of 2.6 muM. Elevated PIP2 levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single channel conductance and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.

Assay Drug Dev Technol. 2009 Nov 18;
Zhang JY, Kowal DM, Nawoschik SP, Dunlop J, Pausch MH, Peri R

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular Calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced Calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.

J Immunol. 2009 Dec 1; 183(11): 7497-7504
Lee SH, Park DW, Park SC, Park YK, Hong SY, Kim JR, Lee CH, Baek SH

Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a Calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.

J Immunol. 2009 Dec 1; 183(11): 7352-7361
Carrizosa E, Gomez TS, Labno CM, Klos Dehring DA, Liu X, Freedman BD, Billadeau DD, Burkhardt JK

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient Calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)gamma1 phosphorylation and Calcium store release, we examined the Calcium signaling pathway in HS1(-/-) T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished Calcium store release, but TCR-dependent PLCgamma1 phosphorylation was intact, indicating that HS1's role in Calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes.

Cell Mol Bioeng. 2009 Sep 1; 2(3): 375-385
Peng HH, Dong C

It has been shown in our previous study that melanoma cells induce junction disassembly in the manner related to phospholipase C-Calcium activation. In light of this observation, we have developed a mathematical model of the signaling pathway and adapted multi-parametric sensitivity analysis (MPSA) to identify important parameters in the model, which examines tumor cell-induced Calcium mobilization in endothelial cells. The objective functions, with respect to individual parameters, were generated for the Calcium mobilization model and MPSA was performed according to the function. The results showed that sarco/endoplasmic reticulum Calcium ATPase was one of the putative key factors in regulating Calcium mobilization. The model is a proof of concept of systemic analysis of a signaling network, and the results may have practical applications in describing how endothelial cells respond to tumor cells. Taken together, we have devised numerical means to macroscopically study roles of Calcium signaling in endothelial cells in contact with metastatic tumor cells.

Am J Physiol Regul Integr Comp Physiol. 2009 Nov 11;
Allen DL, Uyenishi J, Cleary AS, Mehan RS, Lindsay SF, Reed JM

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular Calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the Calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2-10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C2C12 myotubes with the Calcium ionophore A23187. Co-transfection of C2C12 myotubes with a constitutively active calcineurin construct significantly increased while co-transfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Co-transfection with a MEF-2C expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin co-transfection, while co-transfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, co-transfection with a dominant negative form of another calcineurin-activated transcription factor, NFAT, greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C2C12 myotubes. Mutation of the MEF-2 DNA binding sites attenuated while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT. Key words: transcription, exercise, cytokine, treadmill.

Curr Opin Pharmacol. 2009 Nov 6;
Hasbi A, O'Dowd BF, George SR

The repertoire of signal transduction pathways activated by dopamine in brain includes the increase of intracellular Calcium. However the mechanism(s) by which dopamine activated this important second messenger system was/were unknown. Although we showed that activation of the D5 dopamine receptor increased Calcium concentrations, the restricted anatomic distribution of this receptor made this unlikely to be the major mechanism in brain. We have identified novel heteromeric dopamine receptor complexes that are linked to Calcium signaling. The Calcium pathway activated through the D1-D2 receptor heteromer involved coupling to Gq, through phospholipase C and IP(3) receptors to result in a rise in intracellular Calcium. The Calcium rise activated through the D2-D5 receptor heteromer involved a small rise in intracellular Calcium through the Gq pathway that triggered a store-operated channel mediated influx of extracellular Calcium. These novel receptor heteromeric complexes, for the first time, establish the link between dopamine action and rapid Calcium signaling.

J Orthop Res. 2009 Nov 4;
Garcia M, Knight MM

The process of chondrocyte mechanotransduction is poorly understood. However, recent studies suggest the involvement of a purinergic Calcium signaling pathway although the mechanism of ATP release has not been identified. The present study tests the hypothesis that cyclic compression opens hemichannels thereby triggering the release of ATP into the extracellular milieu activating P2 receptors. The well-established chondrocyte-agarose model was utilized enabling chondrocytes to be subjected to a 40-min period of cyclic compression at 0-15% strain and 1 Hz. The opening of hemichannels was determined using Lucifer yellow (LY) incorporation and fluorescence microscopy, whereas the release of ATP into the surrounding media was quantified using the luciferin-luciferase assay. Results indicated that cyclic compression activated hemichannels such that the percentage of cells showing LY incorporation increased from 50 to 70%. This was associated with a sevenfold increase in the release of ATP. Both LY incorporation and ATP release in response to mechanical loading were blocked by the hemichannel inhibitor, flufenamic acid. Treatment with apyrase or P2 receptor antagonists, suramin or oxidated-ATP, did not prevent the mechanically induced response. In conclusion, mechanical loading triggers release of ATP via hemichannels. Hence, this study provides the first evidence of hemichannel involvement in chondrocyte mechanobiology. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Neuropsychopharmacology. 2009 Nov 4;
Wang J, Li MD

Twin and family studies reveal a significant genetic contribution to the risk of smoking initiation and progression (SI/P), nicotine dependence (ND), and smoking cessation (SC). Further, numerous genes have been implicated in these smoking-related behaviors, especially for ND. However, no study has presented a comprehensive and systematic view of the genetic factors associated with these important smoking-related phenotypes. By reviewing the literature on these behaviors, we identified 16, 99, and 75 genes that have been associated with SI/P, ND, and SC, respectively. We then determined whether these genes were enriched in pathways important in the neuronal and brain functions underlying addiction. We identified 9, 21, and 13 pathways enriched in the genes associated with SI/P, ND, and SC, respectively. Among these pathways, four were common to all of the three phenotypes, that is, Calcium signaling, cAMP-mediated signaling, dopamine receptor signaling, and G-protein-coupled receptor signaling. Further, we found that serotonin receptor signaling and tryptophan metabolism pathways were shared by SI/P and ND, tight junction signaling pathway was shared by SI/P and SC, and gap junction, neurotrophin/TRK signaling, synaptic long-term potentiation, and tyrosine metabolism were shared between ND and SC. Together, these findings show significant genetic overlap among these three related phenotypes. Although identification of susceptibility genes for smoking-related behaviors is still in an early stage, the approach used in this study has the potential to overcome the hurdles caused by factors such as genetic heterogeneity and small sample size, and thus should yield greater insights into the genetic mechanisms underlying these complex phenotypes.Neuropsychopharmacology advance online publication, 4 November 2009; doi:10.1038/npp.2009.178.

Toxicol Sci. 2009 Nov 3;
Son YO, Lee JC, Hitron JA, Pan J, Zhang Z, Shi X

Cadmium is a toxic heavy metal and has been widely used in industry. The skin is an important target for this metal. The mechanisms by which cadmium leads to damage to the skin are unclear at present. The aims of this study were to examine whether cadmium induces apoptosis in mouse skin epidermal cell line, JB6 Cl41 cells, and to investigate the cellular mechanisms by which cadmium causes cytotoxicity in the cells. The present study showed that cadmium induced cell death by apoptosis in a dose-dependent manner, as proven by the appearance of cell shrinkage, the increase of Annexin V-positively stained cells, and the formation of nuclear DNA ladders. Cadmium-induced apoptosis involved a mitochondria-mediated mechanism but not caspase-dependent pathway, in that the critical apoptotic events induced by cadmium, such as the decrease of Bcl-2/Bcl-xL, the increase of GADD45alpha, and the nuclear translocation of AIF, were not affected by the inhibition of executive caspases. In contrast, blockage of p53 and JNK by pharmacological inhibitors or si-RNA transfection suppressed the cadmium-induced apoptosis with the concomitant inhibition of anti-apoptotic Bcl-2 family proteins and GADD45alpha, respectively. Further, the activation of p53 and JNK and their downstream proteins in cadmium-exposed cells were inhibited by individual treatment with catalase and Bapta-AM. These results suggest that cadmium induces apoptosis via the activation of JNK- and p53-mediated signaling, where Calcium ion and hydrogen peroxide act as the pivotal mediators of the apoptotic signaling.

J Biol Chem. 2009 Nov 3;
Yang Y, Wu X, Gui P, Wu J, Sheng JZ, Ling S, Braun AP, Davis GE, Davis MJ

Large conductance, Calcium-activated K+ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-Src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca2+ and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca2+-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca2+ concentrations >/= 1 muM. Overexpression of human c-Src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca2+ sensitivity, whereas overexpression of catalytically inactive c-Src blocks integrin-induced potentiation. However, neither integrin activation nor c-Src overexpression potentiates current in BK channels containing a point mutation at Tyr766. Biochemical tests confirm the critical importance of residue Tyr766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-Src phosphorylation of the channel alpha-subunit at Tyr766. The net result is increased current amplitude, enhanced Ca2+ sensitivity and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

J Physiol Biochem. 2009 Jun; 65(2): 125-36
Xia JX, Fan SY, Yan J, Chen F, Li Y, Yu ZP, Hu ZA

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration ([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1-1 microM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of [Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmic reticulum inhibitor thapsigargin (2 pM), [Ca2+]i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca2+-free condition, orexin A failed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 microM) and D609 (10 microM), respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 microM) significantly suppressed the elevation of [Ca2+]i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca2+]i in neurons from deep layers of PFC, which depends on extracellular Ca2+ influx via L-type Ca2+ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.

J Theor Biol. 2009 Oct 31;
Callender HL, Horn MA, Decamp DL, Sternweis PC, Alex Brown H

A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y(6) receptors activated by the ubiquitous signaling nucleotide uridine 5'-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic Calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5'-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty.

Eur J Pharmacol. 2009 Oct 30;
Choi EY, Chang W, Lim S, Song BW, Cha MJ, Kim HJ, Choi E, Jang Y, Chung N, Hwang KC

Statins have recently been shown to produce anti-cardiac hypertrophic effects via the regulation of small GTPases. However, the effects of statins on G protein-mediated cardiac hypertrophy, which is the main pathway of cardiac hypertrophy, have not yet been studied. We sought to evaluate whether statin treatment directly suppresses cardiac hypertrophy through a large G protein-coupled pathway regardless of the regulation of small GTPases. Using neonatal rat cardiomyocytes, we evaluated norepinephrine-induced cardiac hypertrophy for suppressibility of rosuvastatin and the pathways involved by analyzing total protein/DNA content, cell surface area, immunoblotting and RT-PCR for the signal transduction molecule. In a concentration-dependent manner, rosuvastatin inhibited total protein synthesis and downregulated basal and norepinephrine-induced expressions of myosin light chain2 and the c-fos proto-oncogene in cardiomyocytes. Treatment with norepinephrine induced cardiac hypertrophy accompanied by G(h) expression and membrane translocation. Rosuvastatin inhibited G(h) protein activity in cardiomyocytes by inhibiting basal and norepinephrine-stimulated mRNA transcription, protein expression and membrane translocation; however, norepinephrine-stimulated G(q) protein expression was not inhibited. In addition, the norepinephrine-stimulated protein kinase C (PKC)-mitogen-activated protein kinase (MEK 1,2)-extracellular signal-regulated kinases (ERKs) signaling cascade was inhibited by pretreatment with rosuvastatin. Rosuvastatin treatment also helped maintain expression levels of SERCA2a and intracellular Calcium concentration. G(h) protein is a novel target of statins in myocardial hypertrophy, and statin treatment may directly suppress cardiac hypertrophy through a large G(h) protein-coupled pathway regardless of the regulation of small GTPases.

Mol Cell Biochem. 2009 Nov 1;
Orellano EA, Rivera OJ, Chevres M, Chorna NE, González FA

Apoptosis is a major mechanism for cell death in the nervous system during development. P2X(7) nucleotide receptors are ionotropic ATP receptors that mediate cell death under pathological conditions. We developed an in vitro protocol to investigate the expression and functional responses of P2X(7) nucleotide receptors during retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y neuroblastoma cells. Neuronal differentiation was examined measuring cellular growth arrest and neuritic processes elongation. We found that SH-SY5Y cells treated for 5 days with RA under low serum content exhibited a neuron-like phenotype with neurites extending more than twice the length of the cell body and cell growth arrest. Concurrently, we detected the abolishment of intracellular-free Calcium mobilization and the down-regulation of P2X(7) nucleotide receptor protein expression that protected differentiated cells from neuronal cell death and reduced caspase-3 cleavage-induced by P2X(7) nucleotide receptor agonist. The role of P2X(7) nucleotide receptors in neuronal death was established by selectively antagonizing the receptor with KN-62 prior to its activation. We assessed the involvement of protein kinases and found that p38 signaling was activated in undifferentiated after nucleotide stimulation, but abolished by the differentiating RA pretreatment. Importantly, P2X(7) receptor-induced caspase-3 cleavage was blocked by the p38 protein kinase specific inhibitor PD169316. Taken together, our results suggest that RA treatment of human SH-SY5Y cells leads to decreased P2X(7) nucleotide receptor protein expression thus protecting differentiated cells from extracellular nucleotide-induced neuronal death, and p38 signaling pathway is critically involved in this protection of RA-differentiated cells.

Eur J Pharmacol. 2009 Oct 30;
Kammermeier PJ

Recent studies indicate that the intracellular C-terminus of Group I metabotropic glutamate receptors (mGlu(1) and mGlu(5) receptor) is important in G protein coupling. To determine the necessity of the C-tail, a deletion mutant of mGlu(1) receptor was constructed, which included the first 840 amino acids of the rat mGlu(1a) receptor (mGlu(1)-dCT). G protein coupling of the receptors was assessed by measuring glutamate mediated inhibition of native Calcium currents when each receptor was expressed in isolated sympathetic neurons from the rat superior cervical ganglion. Wild type mGlu(1) receptor activates both the Galpha(i/o) and Galpha(q/11) protein families. Each pathway can be detected in superior cervical ganglion neurons as voltage dependent and voltage independent inhibition of the Calcium currents, respectively. While wild type mGlu(1) receptor gave rise to a strong, mixed voltage dependent and independent Calcium current inhibition, mGlu(1)-dCT exhibited a weaker inhibition that was strongly voltage dependent, indicating activation of Galpha(i/o) was predominant. Further, pertussis toxin treatment reduced the inhibition by wild type mGlu(1) receptor to a smaller, voltage independent inhibition as expected, but completely abolished signaling through mGlu(1)-dCT. Finally, to test whether mGlu(1)-dCT could produce any activation of Galpha(q/11), inhibition of the native superior cervical ganglion M-type potassium currents was examined. M-channels, inhibited by PIP(2) depletion, were strongly inhibited by glutamate in cells expressing wild type mGlu(1) receptor, but no inhibition was detectable in neurons expressing mGlu(1)-dCT. These data indicate that C-terminal deletion of mGlu(1) receptor selectively abolishes Galpha(q/11) coupling.

Endocrinology. 2009 Oct 28;
Constantin S, Jasoni CL, Wadas B, Herbison AE

Multiple factors regulate the activity of the GnRH neurons responsible for controlling fertility. Foremost among neuronal inputs to GnRH neurons are those using the amino acids glutamate and gamma-aminobutyric acid (GABA). The present study used a GnRH-Pericam transgenic mouse line, enabling live cell imaging of intracellular Calcium concentrations ([Ca(2+)]i) to evaluate the effects of glutamate and GABA signaling on [Ca(2+)]i in peripubertal and adult mouse GnRH neurons. Activation of GABAA, N-methyl-D-aspartate, or alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid (AMPA) receptors was found to evoke an increase in [Ca(2+)]i, in subpopulations of GnRH neurons. Approximately 70% of GnRH neurons responded to GABA, regardless of postnatal age or sex. Many fewer ( approximately 20%) GnRH neurons responded to N-methyl-D-aspartate, and this was not influenced by postnatal age or sex. In contrast, about 65% of adult male and female GnRH neurons responded to AMPA compared with about 14% of male and female peripubertal mice (P < 0.05). The mechanisms underlying the ability of GABA and AMPA to increase [Ca(2+)]i in adult GnRH neurons were evaluated pharmacologically. Both GABA and AMPA were found to evoke [Ca(2+)]i increases through a Calcium-induced Calcium release mechanism involving internal Calcium stores and inositol-1,4,5-trisphosphate receptors. For GABA, the initial increase in [Ca(2+)]i originated from GABAA receptor-mediated activation of L-type voltage-gated Calcium channels, whereas for AMPA this appeared to involve direct Calcium entry through the AMPA receptor. These observations show that all of the principal amino acid receptors are able to control [Ca(2+)]i in GnRH neurons but that they do so in a postnatal age- and intracellular pathway-specific manner.

BMC Genomics. 2009; 10(1): 499
Costa JL, Eijk PP, van de Wiel MA, Ten Berge D, Schmitt F, Narvaez CJ, Welsh J, Ylstra B

ABSTRACT: BACKGROUND: The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has strong anti-proliferative effects, yet the molecular mechanisms underneath this effect remain unclear. In contrast, the molecular mechanism of 1,25D for the regulation of Calcium homeostasis has principally been resolved, demonstrating a pivotal role for the vitamin D receptor (VDR). RESULTS: We first addressed the question whether the anti-proliferative effects of 1,25D are influenced by VDR. Knockdown of VDR by siRNA did not affect the anti-proliferative effects of 1,25D in MCF7 breast cancer cells. This unanticipated finding led us to take an alternative approach using genome wide screens to study the molecular mechanisms of 1,25D in proliferation. For that purpose, four independently developed and stable 1,25D resistant MCF7 cell lines were analyzed. Array CGH identified a copy number alteration in a region of 13.5 Mb at chromosome 11q13.4-14.1 common to all four 1,25D resistant cell lines. Expression arrays revealed that no single gene was differentially expressed between the sensitive and resistant cells, but multiple membrane receptor signaling pathways were altered in the 1,25D resistant cell lines. Importantly, in the genome wide experiments neither VDR, CYP24A1 nor other known vitamin D signaling pathway genes were associated with 1,25D resistance. CONCLUSION: In conclusion, siRNA and genome wide studies both suggest that the anti-proliferative effects of 1,25D in MCF7 breast tumor cell lines do not rely on classical Vitamin D pathway per se.

Endocr J. 2009 Oct 23;
Hayashi C, Iino K, Oki Y, Matsushita F, Yamashita M, Yogo K, Sasaki S, Kumada T, Nakamura H

Cytoplasmic Calcium ([Ca(2+)](i)) provided through voltage-dependent Ca(2+)channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca(2+)]i supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca(2+)]i supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10(-11) M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10muM 2APB by 51.8% (6.71 +/- 0.97 vs. 3.23 +/- 0.05 ng/mL/4hours; p<0.05). Furthermore, 2APB significantly decreased the 10(-11) M ACTH-stimulated [Ca(2+)](i). ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10(-13) M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca(2+) from the ER or the subsequent store-operated Ca(2+) entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca(2+) supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca(2+)](i) used for steroidogenesis may be derived from a 2APBsensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.